ScRNA-seq of pluripotent stem cell derived human intestinal organoids grown in an alginate matrix
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ABSTRACT: Here, we used single-cell RNA-sequencing (scRNA-seq) to profile pluripotent stem cell derived human intestinal organoids (HIOs) grown in an alginate matrix after 3, 7, and 14 days of in vitro growth. Samples were grown in minigut media supplemented with either ENR or EGF.
Project description:The development of human pluripotent stem cell (hPSC)- derived small intestinal organoids (HIOs) has led to an improved understanding of human intestinal development and physiology. HIOs generated using directed differentiation lack some cellular populations found in the native organ, including vasculature. We performed single cell RNA sequencing (scRNA-seq) on approximately 13,000 cells at various timepoints (0, 3, 7, and 14 days) across HIO in vitro development and observed a transient population of endothelial-like cells (ECs) present within HIOs early during differentiation. Our data demonstrate that EC-like cells fail to be robustly maintained in long term culture. Here, we have developed a new directed differentiation approach to enhance co-differentiation and maintenance of ECs within HIOs, leading to the development of vascularized HIOs (vHIOs). scRNAseq was used to compare vHIOs to control HIOs after 59d months in culture.
Project description:Here, we used single cell RNA-sequencing (scRNA-seq) to profile pluripotent stem cell derived human intestinal organoids (HIOs) grown in matrigel or a non-adhesive alginate hydrogel after 28 days of in vitro growth. Additionally, we used scRNA-seq to profile HIOs derived in the presence of Neuregulin 1 (NRG1) and/or EGF after 40 days of in vitro growth.
Project description:Here, we used single-cell RNA-sequencing (scRNA-seq) to profile pluripotent stem cell-derived human intestinal organoids (HIOs) grown in media comprised of minigut media + varying concentrations of Epiregulin (EREG) 1 ng/ml, 10 ng/ml, 100 ng/ml after 28 days of in vitro growth.
Project description:In vitro human pluripotent stem cell derived intestinal organoids (HIOs) are immature and lack for diverse differentiated secretory cell types. We would like to test the hypothesis whether addition of a mesenchyme secreting ligand which is depleted in canonical organoid culture media could increase the maturity and secretory cell type diversity in HIOs in vitro. To do this, we adapted the directed differentiation protocol of HIOs by growing HIOs in media with EGF, NOGGIN, R-spondin-1 (ENR) for 30 days, isolated epithelial cells with dispase, recovered them with adult intestinal enteroid media with Wnt-3A (WENR). Then we introduced the mesenchyme secreting ligand NRG1 to the established enteroid culture (WENR+NRG1) and compared them to the enteroids grown in control condition (WENR).
Project description:Here, we used single cell RNA-sequencing (scRNA-seq) to profile pluripotent stem cell derived human definitive endoderm and intestinal organoids (HIOs) at several timepoints of in vitro growth (7, 14, and 28 days) and after in vivo growth beneath the kidney capsule of a murine host (4 and 8 wks post-transplant). Additionally, we profiled HIOs grown in a non-adhesive alginate hydrogel and also CDX2 knockout HIOs. In order to benchmark the organoid cultures, we used scRNA-seq to profile primary human fetal esophagus (14.3 pcw, 16.7 pcw), stomach (6.7, 14.3, and 16.7 pcw), liver (14.4 pcw), small intestine ( 11.4 and 14.4 pcw) and colon (11.4, 14.4, and 18.9 pcw). Diverse cell lineages were captured across all tissues profiled, including: epithelium, mesenchyme, neurons, endothelium, and immune lineages.
Project description:Here, we used single-cell RNA-sequencing (scRNA-seq) to profile pluripotent stem cell derived human intestinal organoids (HIOs) grown in suspension culture after 28 days of in vitro growth. Grown in minigut media supplemented with EGF.
Project description:Underdeveloped lungs are a primary cause of morbidity and mortality in premature infants, but our ability to help these patients by speeding up lung development are hindered by a lack of understanding of human lung developmental biology. Here, we performed single cell RNA sequencing of the human fetal lung from samples spanning from 11.5 weeks gestation to 21 weeks gestation from the distal lung, middle airways, and the tracheal epithelium. The primary goal of this experiment was to define fetal cell states to serve as a gold standard for pluripotent stem cell-derived lung cells and tissues, and to identify potential signaling pathways that drive differentiation of lung progenitor cells to mature cell types. Additionally, we generated bud tip progenitor organoids from 12 week human fetal lung bud tip progenitors. We show that treatment of bud tip progenitor organoids with a short pulse of dual SMAD activation (BMP4+TGFb1) led to the upregulation of lung basal cell markers, a cell type that serves as a critical stem cell for the adult airway, and that further treatment with dual SMAD inhibition leads to the generation of airway-like organoids containing differentiated cell types of the adult airway, including basal stem cells.
Project description:Molecular characterization of tissue-resident memory T cells cultured with or without donor-matched adult stem cell-derived intestinal organoids. Blood derived immune cells were also isolated and cultivated with autologous intestinal organoids for comparison and characterization. All conditions were derived from 3 human individuals and all samples were sequenced after 24h of in vitro culture. Data provides insights on circulating and tissue-resident immune cell populations, how these differentially interact with the epithelium and how these interactions shape both immune and epithelial cell states.
Project description:We set up a 3D model based on iPSCs derived from patients with familial forms of Alzheimer’s disease (AD) and healthy non-demented control. We created cerebral organoids (COs), verified their ability to mimic AD in vitro, and used it to explore early events and the progression of AD pathogenesis. Our data reveal that despite similar expression of cell-type-specific genes during CO maturation in vitro, AD-iPSCs derived COs show limited tissue patterning and altered cellular development. These findings complement unique single-cell sequencing data of AD-iPSCs derived COs confirming this observation and uncovering that a sub-set of neurons in AD-iPSCs likely differentiates prematurely while at the same time retaining the expression of progenitor marker PAX6.